Preparation of ammonium salt of hypocholesteremic fermentation product

ABSTRACT

Substances isolated after cultivation of a microorganism belonging to the genus Aspergillus in a culture medium comprise compounds which have structures: ##STR1## Together with salts and esters of the carboxylic acid, these compounds form a class of highly active hypocholesteremic and hypolipemic medicaments.

SUMMARY OF THE INVENTION

This is a division of copending application Ser. No. 159,983, filed June16, 1980 by Albers-Schonberg, Monaghan, Alberts and Hoffman, which is acontinuation-in-part of copending application Ser. No. 114,459, filedJan. 23, 1980, abandoned, which in turn is a continuation-in-part ofapplication Ser. No. 048,946, filed June 15, 1979, U.S. Pat. No.4,231,938.

This invention relates to hypocholesteremic products from thecultivation of a microfungus of the genus Aspergillus. Morespecifically, it relates to compounds of the formulae: ##STR2## as wellas pharmaceutically acceptable salts and lower alkyl and substitutedalkyl esters of the carboxylic acids in which the possible substituentis phenyl, dimethylamino or acetylamino. The invention also relates to aprocess of cultivating the microfungus and isolating from the medium ahypocholesteremic compound of the above structures. These new compoundshave excellent properties of inhibiting cholesterol biosynthesis and areuseful against hypercholesteremia and hyperlipemia.

BACKGROUND OF THE INVENTION

Because of the possible connection between high blood cholesterol andatherosclerosis, many efforts have been made to find ways and substanceswhich would reduce the cholesterol in the mammalian body. One of theseways is to inhibit in mammals the body's ability to synthesizecholesterol.

Recently, Endo et al., described (U.S. Pat. Nos. 4,049,495 and3,983,140) a fermentation product obtained by cultivation of amicroorganism of the genus Penicillium and isolation from the medium.They called it ML 236 B and determined its structure together with tworelated compounds 236 A and 236 C. Its structure, under the namecompactin, was also determined by A. G. Brown, T. C. Smale, T. J. King,J. Chem. Soc. (Perkin I) 1165 (1975). This compound has been found to bean inhibitor, in vivo, of the biosynthesis of cholesterol.

DESCRIPTION OF THE INVENTION

We have found that unexpectedly, the cultivation of a microorganism verydifferent from that employed by Endo, a microfungus of the genusAspergillus, produces new substances that are also very potentinhibitors of the biosynthesis of cholesterol in mammals. We havefurther found that these substances comprise principally the newcompounds I and II, of the above structures, accompanied by only tracesof other compounds. These new compounds are much more potent inhibitorsof cholesterol synthesis in vivo than is the compound, ML236B describedby Endo.

The pharmaceutically acceptable salts of this invention include thoseformed from cations such as sodium, potassium, aluminum, calcium,lithium, magnesium, zinc, ammonia, ethylenediamine, N-methylglucamine,lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,1-p-chlorobenzyl-2-pyrrolidine-1'-yl-methylbenzimidazole, diethylamine,piperazine, tris-(hydroxymethyl)aminomethane, and tetramethylammonium.

The compounds of this invention are highly useful asantihypercholesteremic agents for the treatment of atherosclerosis,hyperlipemia and like diseases in humans. They may be administeredorally or parenterally in the form of a capsule, a tablet, an injectablepreparation or the like. It is usually desirable to use the oral route.Doses may be varied, depending on the age, severity, body weight andother conditions of human patients but daily dosage for adults is withina range of from about 2 mg. to 2000 mg. (preferably 2 to 100 mg) whichmay be given in two to four divided doses. Higher doses may be favorablyemployed as required.

The compounds of this invention also have useful antifungal activities.For example, they may be used to control strains of Penicillium sp.,Aspergillus niger, Cladosporium sp., Cochiobolus miyabeanus andHelminthosporium cynodnotis. For those utilities they are admixed withsuitable formulating agents, powders, emulsifying agents or solventssuch as aqueous ethanol and sprayed or dusted on the plants to beprotected.

In another aspect of this invention, it relates to a process forproducing the compounds of this invention which comprises cultivating amicroorganism belonging to the genus Aspergillus and then recoveringsaid compounds of this invention from the cultured broth. Based upontaxonomic studies, this Aspergillus, isolated and identified as ahitherto undescribed microorganism, has been designated MF-4833 in theculture collection of Merck and Co., Inc., Rahway, N.J. and a culturethereof has been placed on permanent deposit with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and hasbeen assigned accession number ATCC 20541. Another sample, of a similarorganism, designated MF-4845 in the Merck culture collection, haslikewise been placed on deposit and has been given the accession numberATCC 20542. The latter organism is the one giving the better yield.Although the use of these is described in connection with the process ofthis invention, other organisms of the genus Aspergillus includingmutants of the above ones are also capable of producing these novelcompounds and their use is contemplated in carrying out the process ofthis invention.

The morphological characteristics of the microorganisms MF-4833 andMF-4845 have been found to be those of the genus Aspergillus. Using thecriteria specified in the standard authority "Manual of the Aspergilli",Charles Thom and Kenneth B. Rasper, published by the Williams andWilkins Company, Baltimore, Md., 1945, and by comparison with knownspecies, it has been determined that both strains are Aspergillusterreus.

The culture of these organisms to produce the novel compounds is carriedout in aqueous media such as those employed for the production of otherfermentation products. Such media contain sources of carbon, nitrogenand inorganic salts assimilable by the microorganism.

In general, carbohydrates such as sugars, for example, glucose,fructose, maltose, sucrose, xylose, mannitol and the like and starchessuch as grains, for example, oats, ryes, cornstarch, corn meal annd thelike can be used either alone or in combination as sources ofassimilable carbon in the nutrient medium. The exact quantity of thecarbohydrate source or sources utilized in the medium depend in partupon the other ingredients of the medium but, in general, the amount ofcarbohydrate usually varies between about 1% and 6% by weight of themedium. These carbon sources can be used individually, or several suchcarbon sources may be combined in the medium. In general, manyproteinaceous materials may be used as nitrogen sources in thefermentation process. Suitable nitrogen sources include for example,yeast hydrolysates, primary yeast, soybean meal, cottonseed flour,hydrolysates of casein, corn steep liquor, distiller's solubles ortomato paste and the like. The sources of nitrogen either alone or incombination, are used in amounts ranging from about 0.2% to 6% by weightof the aqueous medium.

Among the nutrient inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, ammonium, calcium, phosphate, sulfate, chloride, carbonate,and like ions. Also included are trace metals such as cobalt, manganese,iron and magnesium.

It should be noted that the media described in the Examples are merelyillustrative of the wide variety of media which may be employed, and arenot intended to be limitative. Specifically, the carbon sources used inthe culture media to produce the novel compounds of this inventionincluded dextrose, dextrin, oat flour, oatmeal, molasses, citrate,soybean, oil, glycerol, malt extract, cod liver oil, starch, ethanol,figs, sodium ascorbate and lard oil. Included as nitrogen sources werepeptonized milk, autolyzed yeast, yeast RNA, tomato paste, casein,primary yeast, peanut meal, distillers solubles, corn steep liquor,soybean meal, corn meal, NZ amine, beef extract, asparagine, cottonseedmeal and ammonium sulfate. The major ionic components were CaCO₃, KH₂PO₄, MgSO₄.7H₂ O and NaCl and small amounts of COCl₂.6H₂ O and traces ofFe, Mn, Mo, B and Cu were also present.

The fermentation is carried out at temperatures ranging from about 20°to 37° C.; however, for optimum results it is preferable to conduct thefermentation at temperatures of from about 22° to 30° C. The pH of thenutrient media suitable for growing the Aspergillus culture andproducing the novel compounds can vary from about 6.0 to 8.0.

Although the novel compounds are produced by both surface and submergedculture, it is preferred to carry out the fermentation in the submergedstate. A small scale fermentation is conveniently carried out byinoculating a suitable nutrient medium with the Aspergillus culture and,after transfer to a production medium, permitting the fermentation toproceed at a constant temperature of about 28° C. on a shaker forseveral days.

The fermentation is initiated in a sterilized flask of medium via one ormore stages of seed development. The nutrient medium for the seed stagemay be any suitable combination of carbon and nitrogen sources. The seedflask is shaken in a constant temperature chamber at about 28° C. for 2days, or until growth is satisfactory, and some of the resulting growthis used to inoculate either a second stage seed or the productionmedium. Intermediate stage seed flasks, when used, are developed inessentially the same manner, that is, part of the contents of the flaskfrom the last seed stage are used to inoculate the production medium.The inoculated flasks are skaken at a constant temperature for severaldays, and at the end of the incubation period the contents of the flasksare centrifuged or filtered.

For large scale work, it is preferable to conduct the fermentation insuitable tanks provided with an agitator and a means of aerating thefermentation medium. According to this method, the nutrient medium ismade up in the tank and sterilized by heating at temperatures of up toabout 120° C. Upon cooling, the sterilized medium is inoculated with apreviously grown seed of the producing culture, and the fermentation ispermitted to proceed for a period of time as, for example, from 3 to 5days while agitating and/or aerating the nutrient medium and maintainingthe temperature at about 28° C. This method of producing the novelcompounds is particularly suited for the preparation of largequantities.

The compounds are conveniently isolated from the fermentation broth asthe lactone (I), or as salts of Compound II.

Compound I can be hydrolyzed with bases such as NaOH to yield the saltssuch as the sodium salt of Compound II. The use of bases with otherpharmaceutically acceptable cations affords salts of these cations.Careful acidification of the salts affords the hydroxy acid II. Thehydroxy acid II can be converted to Compound I at acidic pH. TreatingCompound I under acidic or basic catalysis with methanol, ethanol,propanol, or butanol or with phenyl, dimethylamino, or acetylaminoalkanols yields the corresponding esters of Compound II which also forma part of this invention.

Compound II can be conveniently isolated without need of chromatography,in the form of the ammonium salt. The process comprises acidifying wholebroth, preferably with phosphoric acid and preferably to about pH 5;extracting the acidified broth with a water-immiscible, inert, organicsolvent, preferably ethyl acetate; extracting the organic solventextract with aqueous alkali, such as 0.1 to 0.5 N sodium hydroxide;reacidification to pH 5 with phosphoric acid; extracting the acidifiedaqueous extract with a water-immiscible, inert, organic solventpreferably a mixture of n-hexane/ethyl acetate and preferably about a2:1 (v/v) mixture; drying the extract; and bubbling in gaseous ammoniacausing precipitation of the ammonium salt. This isolation is convenientand is much more adapted to commercial use than is chromatography.Furthermore, salts of II are much more active than compound I in vitroin the inhibition of cholesterol biosynthesis and as antifungal agents.Therefore, these salts are one of the especially preferred dosage forms.Preferred salts, in addition to ammonium, include tetramethylammonium,and salts of ethylenediamine, sodium, potassium, calcium,N-methylglucamine, lysine, arginine and ornithine.

The physico-chemical properties of compound I (MSD-803) are summarizedas follows:

    ______________________________________                                        1.    Melting point    170-171°                                        2.    Molecular Weight 404                                                          (mass spectrum)                                                         3.    Formula          C.sub.24 H.sub.36 O.sub.5                                    (found by mass spec-                                                                           404.2555                                                     trometry                                                                      calculated)      404.2563                                               4.    UV Spectrum                                                                   (in acetonitrile):                                                                             Maxima                                                                        230.5 nm with E% 505.7                                                        237.5 nm with E% 576.6                                                        246 nm with E% 395.2                                   ______________________________________                                    

5. ¹³ C NMR chemical shifts.

The spectrum has been recorded in CDCl₃ solution (20.1 mg in 0.35 ml).Chemical shifts are given relative to internal tetramethylsilane at zeroppm; under the experimental conditions the solvent (CDCl₃) signalappears centered at 70.0 ppm. In agreement with mass spectral data 24carbon atoms are observed; their chemical shifts are:

11.5, 13.6, 16.0, 22.6, 24.1, 26.6, 27.2, 30.5, 32.5, 32.8, 35.9, 36.4,37.1, 38.4, 41.3, 62.4, 67.8, 76.4, 128.4, 129.7, 131.7, 133.2, 170.8and 177.2 ppm.

6. ¹ H NMR Spectrum

The spectrum was recorded in CDCl₃ solution and chemical shifts areshown in FIG. 1 in ppm relative to internal tetramethylsilane at zeroppm.

7. IR Spectrum

The infra red spectrum was recorded in a KBr pellet preparation of asample. It is shown in FIG. 2.

8. Optical rotation.

The specific optical rotation [α]_(D) ²⁵ =320.7° has been determined ona solution of 5.30 mg/ml CH₃ CN. This value has been obtained bymeasuring at the sodium-D-line wave length.

On the basis of these and other data, the structure of the product isbelieved, with a considerable degree of certainty, to have the stereochemical structure: ##STR3## The corresponding hydroxy acid compound II,has the structure: ##STR4## The absolute configuration of the centers ofassymetry in these molecules has been determined from X-ray diffractionpatterns.

This invention can be illustrated by the following examples.

EXAMPLE 1 PREPARATION OF COMPOUNDS I AND II

A. Fermentation

A tube of lyophilized culture MF-4833 is opened aseptically and thecontents suspended in an unbaffled 250 ml Erlenmeyer flask (seed flask)containing approximately 20 ml of medium A. Medium A has the followingcomposition:

    ______________________________________                                        Medium A                                                                      Corn steep liquor       10 g                                                  Tomato paste            80 g                                                  Oatmeal                 20 g                                                  Glucose                 20 g                                                  Trace Element Mix No. 2 20 ml                                                 Distilled water         1000 ml                                               pH 6 . 8 with NaOH                                                            Trace Element Mix No. 2                                                       FeSO.sub.4 . 7H.sub.2 O 1000 mg                                               MnSO.sub.4 . 4H.sub.2 O 1000 mg                                               CuCl.sub.2 . 2H.sub.2 O 25 mg                                                 CaCl.sub.2 . 2H.sub.2 O 100 mg                                                H.sub.3 BO.sub.3 .      56 mg                                                 (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 . 4H.sub.2 O                                                       19 mg                                                 ZnSO.sub.4 . 7H.sub.2 O 200 mg                                                Distilled Deionized Water                                                                             1000 ml                                               ______________________________________                                    

The inoculated flask is incubated for 48 hours at 28° C. on a 220 rpmshaker (2 inch throw). Two unbaffled 2 liter Erlenmeyer flasks eachcontaining 500 ml of medium B are then each inoculated with 10 ml perflask of the growth from the seed flask. Medium B has the followingcomposition:

    ______________________________________                                         Medium B                                                                     ______________________________________                                        Tomato paste           20 g                                                   Primary yeast          10 g                                                   CPC Starch             20 g                                                   CoCl.sub.2 . 6H.sub.2 O                                                                              5 mg                                                   Distilled water        1000 ml                                                pH 7.2-7.4 with NaOH                                                          ______________________________________                                    

These two inoculated flasks are incubated for 96 hours at 28°. One flaskis incubated without agitation. The other flask is incubated on a 150rpm shaker (2" throw). After 96 hours, the contents of each flask areset aside for isolation of the product.

B. Isolation of Compound I

The whole broth is centrifuged for 20-30 min. Solids are saved forextraction. The supernatant liquid (pH 6-8) is charged to a 950 mlbottle and 150 ml XAD-2 resin is added. Using an automatic Extractor,operating on a preset schedule, the mixture is stirred for 2 hours. Thespent broth is then siphoned off and discarded. The resin is washedtwice with 200 ml of deionized water and the washes were discarded.There then is added a charge of 300 ml of mixed solvent:isopropanol-ethyl acetate-dichloromethane 25-45-30. The mixture isstirred two hours. The solvent-resin slurry is filtered on a Buchner orsintered glass funnel and the resin is discarded. The broth solids arestirred with 100 ml acetone for 1/2 hour. The mixture is thencentrifuged and the supernatant liquor is decanted. The combinedfiltrate and decantate are concentrated to 15 ml.

C. Testing of Compound I

The filtrates were tested as inhibitors of HMG-CoA reductase enzyme bythe method described by Beg, Stonik, and Brewer (1977 FEBS Letters 80123 to 129) using enzymes prepared as described by Kleinsek, Rangathamand Porter (1977 Proc. Nat. Acad. Sci. 74 1431 to 1435). The positivetest (over 90% inhibition at 20 micrograms per milliliter-an IC₅₀ of 2.3micrograms per milliliter) indicated the presence of a very potentinhibitor of sterol synthesis acting at the HMG-CoA reductase level.

EXAMPLE 2 PREPARATION OF COMPOUNDS I AND II

A. Fermentation

A tube of lyophilized culture of an Aspergillus sp. MF-4833 is openedaseptically and the contents suspended in an unbaffled 250 ml Erlenmeyerflask (seed flask No. 1) containing 40 ml of medium C. Medium C has thefollowing compositions:

    ______________________________________                                        Medium C                                                                      Corn steep liquor     5 g                                                     Tomato paste          40 g                                                    Oatmeal               10 g                                                    Glucose               10 g                                                    Trace element                                                                 Mix No. 2             10 ml                                                   Distilled water       1000 ml                                                 pH 6.8 with NaOH                                                              ______________________________________                                    

This inoculated flask is incubated for 24 hours at 28° C. on a 220 rpmshaker (2 inch throw) for 24 hours. Eight more unbaffled 250 mlErlenmeyer flasks (No. 2 seed flask) each containing 40 ml of medium Care then each inoculated with 2 ml per flask of the growth from seedflask No. 1. These eight No. 2 seed flasks are incubated for 24 hours at28° C. on a 220 rpm shaker (2 inch throw). Twenty, two liter unbaffledErlenmeyer flasks, containing 500 ml of medium B are then eachinoculated with 14 ml per flask of the combined growth of the eight No.2 seed flasks. These twenty flasks are incubated at 28° C., withoutagitation for 11 days. After 11 days incubation, the contents of thesetwenty flasks are pooled.

B. Extraction

10.2 liters of whole broth, pH 6.0, was blended in a Waring blender tobreak up the heavy mycelial pads, centrifuged and the clear supernatantdecanted. After filtration the 10 liters of filtrate was extracted with3 liters of ethyl acetate, yielding 1820 ml of clear extract. A secondextraction with 3 liters of ethyl acetate yielded 3350 ml of clearextract. The broth solids were extracted by stirring one hour with 2liters of methanol and filtering to yield 2100 ml of filtrate.

Aliquots of these extracts were dried and sent for assay by theprocedure of Example 1(C), with the following results:

    ______________________________________                                        Extract                                                                       Volume (ml)                                                                             Total Solids (mg)                                                                           Total Units of Activity                               ______________________________________                                        1820      1133          1,496,695                                             3350      787             314,900                                             2100      13.15         1,144,067                                             ______________________________________                                    

C. Gel Filtration

The total solids obtained from the first two extracts in Example 2 (B)were combined, dissolved in methanol and filtered to remove insolublesolids. The 30 ml of filtrate was loaded onto a gel filtration column(2.5 cm×200 cm, 980 ml) packed with Sephadex LH-20 and the samplefractionated according to molecular size using methanol as solvent. Withrefractive index and U.V. recordings as guides, the best fractions wereidentified by bioassay.

    ______________________________________                                        Total Solids (mg)                                                                           Total Units of Activity                                         ______________________________________                                        Fraction 1 - 89                                                                             106,271                                                         Fraction 2 - 278                                                                            1,099,680                                                       Fraction 3 - 779                                                                            210,357                                                         ______________________________________                                    

D. Separation and Purification

A sample from Fraction 2 above was pre-filtered through a 1-gram bed ofWaters Bondapak C18/Porasil B and eluted with five volumes of methanol.The methanol eluate was concentrated to 0.5 ml. This sample waschromatographed, over several runs, on a Waters μC18 column (3.9 mm×30cm) with methanol: 0.05 M ammonium phosphate, pH 2.9 (75:25), as thedeveloping solvent. Fractions were scanned on a BeckmanSpectrophotometer, and those showing absorption maxima at 236 nm, withshoulders at 229 nm and 245 nm were combined and concentrated underreduced pressure to an aqueous solution. The pH of the concentrate wasadjusted to 6.5 with 2 M potassium hydroxide and the active componentswere extracted with ethyl acetate. The organic layer was dried,concentrated to dryness, and the residue was dissolved in 0.3 mlmethanol. The methanol solution was chromatographed as above andrecycled. Cuts containing earlier eluting component were combined,concentrated to an aqueous solution and extracted with chloroform. Thechloroform residue was taken up in methanol and the solvent evaporatedunder nitrogen. 3.5 mg. of dried product was obtained and identified ashydroxy acid (compound II). Cuts containing the second component werecombined and extracted with chloroform as above. 0.87 Mg of driedproduct was obtained and identified as lactone, (compound I).

EXAMPLE 3 BEST MODE OF FERMENTATION OF MF-4833 TO PRODUCE COMPOUNDS IAND II

A tube of lyophilized culture of an Aspergillus sp. MF-4833 is openedaseptically and the contents suspended in an unbaffled 250 ml Erlenmeyerflask (seed flask) containing 40 ml of medium C. The inoculated flask isincubated for 48 hours at 28° C. on a 220 rpm shaker (2 inch throw).Two, 250 ml unbaffled Erlenmeyer flasks each containing 40 ml of mediumD are then each inoculated with 2 ml per flask of the growth from theseed flask. Medium D has the following composition:

    ______________________________________                                        Medium D                                                                      ______________________________________                                        Lactose                20     g                                               Distillers solubles    15     g                                               Autolyzed yeast        5      g                                               Distilled water        1000   ml                                              ph 7.0 with NaOH                                                              ______________________________________                                    

These two inoculated flasks are incubated for 96 hours at 28° C. on a150 rpm shaker (2 inch throw). After 96 hours incubation the contents ofthese two flasks are submitted for extraction by the procedure describedin Example 2(B). Total production in these flasks is 1450-2000 units/ml.

EXAMPLE 4 PREPARATION OF COMPOUNDS I AND II

A tube of lyophilized culture of an Aspergillus, MF 4845, is openedaseptically and the contents suspended in an unbaffled 250 ml Erlenmeyerflask (seed flask No. 1) containing 40 ml of medium C. The inoculatedflask is incubated for 24-48 hours at 28° C. on a 220 rpm shaker (2 inchthrow). A portion (approx. 0.5 ml) of this flask is then used toinoculate a slant tube containing medium E. Medium E has the followingcomposition:

    ______________________________________                                        Medium E                                                                      ______________________________________                                        Yeast Extract          4      g                                               Malt Extract           10     g                                               Dextrose               4      g                                               Agar                   20     g                                               Distilled Water        1000   ml                                              pH 7.0 with NaOH                                                              ______________________________________                                    

The inoculated slant tube is incubated for 11 days at room temperature.It is then stored at -60° C. for 3-4 months. A portion of the contentsof this slant is then suspended in an unbaffled, 250 ml Erlenmeyer flask(No. 2 seed flask) containing 40 ml of medium C. The inoculated flask isincubated for 24 hours at 28° C. on a 220 rpm shaker (2 inch throw). Sixunbaffled 250 ml Erlenmeyer flasks (No. 3 seed flasks) containing 40 mlof medium C are then each inoculated with 2 ml per flask of the growthfrom the No. 2 seed flask. These six inoculated flasks are incubated for48 hours at 28° C. on a 220 rpm shaker (2 inch throw). Six unbaffled twoliter Erlenmeyer flasks containing 500 ml of medium F are each theninoculated with the contents of No. 3 seed flask. Medium F has thefollowing composition:

    ______________________________________                                        Medium F                                                                      ______________________________________                                        Corn steep liquor      15     g                                               CPC Starch             20     g                                               Corn meal              1      g                                               Soybean meal           4      g                                               Glucose                5      g                                               Soybean oil            2.5    g                                               (NH.sub.4).sub.2 SO.sub.4                                                                            4      g                                               KH.sub.2 PO.sub.4      0.3    g                                               CaCo.sub.3             6      g                                               Distilled Water        1000   ml                                              pH 6.7 with NaOH                                                              ______________________________________                                    

The inoculated flasks are incubated for 11 days without agitation at 28°C. After 11 days broth is delivered for extraction by the procedure ofExample 2(B). Total production in these flasks is 1231 units/ml.

EXAMPLE 5 BEST MODE OF FERMENTATION WITH MF-4845 TO PRODUCE COMPOUNDS IAND II

A tube of lyophilized culture of an Aspergillus, MF-4845, is openedaseptically and the contents suspended in an unbaffled 250 ml Erlenmeyerflask (seed flask) containing 40 ml of medium C. The inoculated flask isincubated for 30 hours at 28° C. on a 220 rpm shaker (2 inch throw). Anunbaffled, 250 ml Erlenmeyer flask containing 40 ml of medium G isinoculated with 2 ml per flask of the growth from the seed flask. MediumG has the following composition:

    ______________________________________                                        Medium G                                                                      ______________________________________                                        Dextrose               45     g                                               Peptonized milk        24     g                                               Autolyzed yeast        2.5    g                                               Polyglycol P2000       2.5    ml                                              Distilled water        1000   ml                                              pH 7.0 with NaOH                                                              ______________________________________                                    

This inoculated flask is incubated for 120 hours at 28° C. on a 220 rpmshaker (2 inch throw). After 120 hours incubation, the contents of theflask is submitted for extraction by the procedure of Example 2 (B).Total production in this flask is 21,500 units/ml.

EXAMPLE 6

A. Large Scale Fermentation with MF-4833 to Produce Compounds I and II

The medium used in each step of the fermentation comprised:

    ______________________________________                                        Corn steep liquor       5      g                                              Tomato paste            40     g                                              Oat Fluor               10     g                                              Glucose                 10     g                                              Trace element solution  10     ml                                             Distilled water         1000   ml                                             ______________________________________                                    

adjusted to pH 6.8 with sodium hydroxide.

The trace element solution comprised:

    ______________________________________                                        FeSO.sub.4 . 7H.sub.2 O                                                                              1      g                                               MnSO.sub.4 . 4H.sub.2 O                                                                              1      g                                               CuCl.sub.2 . 2H.sub.2 O                                                                              25     mg                                              CaCl.sub.2             100    mg                                              H.sub.3 BO.sub.3       56     mg                                              (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 . 4H.sub.2 O                                                      19     mg                                              Zn SO.sub.4 . 7H.sub.2 O                                                                             200    mg                                              distilled water        1      liter                                           ______________________________________                                    

All media were checked for sterility before innoculation with amicroorganism.

To a 250 ml non-baffled Erlenmeyer flask was charged 40 ml of medium andthe contents of one tube of lyophillized organism MF 4833. It was thenshaken for 24 hours at 28° C. on a rotary shaker at 220 rpm. New flaskswere then charged with 40 ml of medium and 1 ml of the first flask'scontents and were shaken an additional 24 hours at 28° C. A 2 literflask was then charged with 400 ml of medium and 10 ml of the secondstage fermentation mixture and this too was shaken for 24 hours at 28°C.

A 200 gallon stainless steel fermentation vat was then charged with 501liters of a medium comprising:

    ______________________________________                                        lactose              2% wt/vol                                                distiller solubles   1.5% wt/vol                                              autolyzed yeast      0.5% wt/vol                                              Polyglycol P2000     0.25% wt/vol                                             ______________________________________                                    

whose pH was adjusted to 7.0. This was sterilized 15 minutes at 121° C.One liter of the third stage above was then charged and the mixture wasincubated at 130 rpm at 28° C. for 96 hours with an air flow of 10 cfm.

B. Isolation of Compound I

About 37.5 lbs. (3/4 bag) of a silicaceous filter aid was added to 110gal. whole broth from the culture of MF-4833 described above and themixture was filtered through an 18-inch filter press. The clarifiedfiltrate, (pH 6.6) was adjusted to pH 4.0 by careful addition of 450 mlof concentrated hydrochloric acid, and extracted by agitation with aboutone-third volume (36 gal.) of ethyl acetate. After separation, the uppersolvent layer was removed, and the water phase again extracted withethyl acetate (38 gal.) in a similar fashion. After separation, the twoextracts were combined and back-washed by agitation with about twelvegallons of water. After separation, the ethyl acetate solution wasconcentrated under vacuum at a temperature below 30° C., first in astirred kettle, and finally in a rotary vacuum evaporator to a residualvolume of slightly less than one gallon.

Approximately 1 gal. (3800 ml) of ethyl acetate concentrate from thepreceding extraction was further concentrated in a rotary evaporator (ca10 mm, 40° C. bath) to a syrup and was then concentrated twice more,after addition of about one liter of methylene chloride in two portions,to free the syrup of polar solvent. The final oil of about 300 ml whichcontained about 250 g of solids by dry weight determination, was made upto about 750 ml with ethyl acetate methylene chloride (30/70; v/v) and200 g of silica gel was added and mixed in to form a slurry. This waslayered over the top of a 14 cm by 36 cm column bed holding 2.5 kg ofthe same silica gel, in about 7.5 l volume, which had been packed as aslurry in the same solvent mixture. Development with the same solventwas continued until 3 liters of effluent was taken off as forerun.

Development with ethyl acetate-methylene chloride (50/50; v/v) wasbegun, taking 800 ml effluent fractions. Twelve fractions were taken,then 100% ethyl acetate elution was begun, and after seven morefractions, 100% acetone elution was begun. Fractions four throughtwenty-four were assayed for bio-activity in the HMG-CoA Reductaseinhibition assay referred to in Example 1. Substantial activity wasfound in fractions 7 through 11. Peak activity was found in fraction 8.It was concentrated to an oil for further purification; dry wt. bysolids determination was 9.0 gm.

Fraction 8 from the silica gel column was triturated with 50 mlmethylene chloride and filtered; the dried filter cake weighed 4.9 gm.The filtrate was charged to a 2-inch I.D. by 1-meter long column filledwith Sephadex LH-20 dextran gel (Pharmacia) swollen and equilibrated inmethylene chloride, and the column was eluted with methylene chloride ata rate of 15 ml/min. Compound I was eluted between 0.64 and 0.81 columnvolumes. Solvent was removed from this peak leaving a slightly brownresidue weighing approximately 0.290 gm. This residue (213 mg) was takenup in 1.5 ml of CH₂ Cl₂ --CH₃ CN (65-35), charged to a prepacked andequilibrated silica gel column (EM LOBAR Size B) and eluted with CH₂ Cl₂--CH₃ CN (65-35) at 5 ml/min. Evaporation of solvent from the peakeluting between 235 and 360 ml of eluant left 121 mg of crystallineproduct, m.p. 155°-160° C. HPLC of this material on a EM RP 18reverse-phase analytical column (E. Merck HIBAR II, Cat. No. 906046)using 0.05 M sodium phosphate pH 3.0-acetonitrile 45-55 as eluant at 2ml/min. showed a characteristic uv absorbing peak at 11 min.

Eighty-two mg of this material was recrystallized from 0.6 ml ofabsolute ethanol, then again from 0.4 ml of the same solvent to afford,after drying over-night in a desiccator over P₂ O₅, 40 mg of whitefeathery crystals. Analytical HPLC on the system described above gave asingle sharp peak at 11 minutes elution time. After furtherrecrystallizations, a melting point of 170°-171° C. was obtained.

The product was identified by spectra, etc., as Compound I. Thismaterial, in the in vitro HMG-CoA reductase test (of Example 1) gave anIC₅₀ of 0.01 micrograms per milliliter.

EXAMPLE 7 Preparation of Compounds I and II

A. Fermentation

A tube of lyophilized culture MF-4845 is opened aseptically and thecontents suspended in an unbaffled 250 ml Erlenmeyer flask (seed flask)containing approximately 10 ml of the Medium which has the followingcomposition:

    ______________________________________                                        Medium                                                                        ______________________________________                                        Corn steep liquor       5      g                                              Tomato paste            40     g                                              Oatmeal                 10     g                                              Glucose                 10     g                                              Trace Element Solution  10     ml                                             Distilled water         1000   ml                                             pH 6.8 with NaOH                                                              ______________________________________                                    

    ______________________________________                                        Trace Element Solution:                                                       ______________________________________                                        FeSO.sub.4 . 7H.sub.2 O 1000   mg                                             MnSO.sub.4 . 4H.sub.2 O 1000   mg                                             CuCl.sub.2 . 2H.sub.2 O 25     mg                                             CaCl.sub.2 . 2H.sub.2 O 100    mg                                             H.sub.3 BO.sub.3        56     mg                                             (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 . 4H.sub.2 O                                                       19     mg                                             ZnSO.sub.4 . 7H.sub.2 O 200    mg                                             Distilled Deionized Water                                                                             1000   mg                                             ______________________________________                                    

The inoculated flask is incubated for 24 hours at 28° C. on a 220 rpmshaker (2 inch throw). An unbaffled 2 liter Erlenmeyer flask containing500 ml of the medium is then inoculated with 10 ml of the first stagefermentation growth from the seed mixture. This too was shaken 24 hoursat 28° C.

A 200 gallon stainless steel fermentation vat was then charged with 485liters of a medium comprising:

    ______________________________________                                        Cerelose             4.5% wt/vol                                              Peptonized milk      2.5% wt/vol                                              Autolyzed yeast      0.25% wt/vol                                             Polyglycol P2000     0.25% vol/vol                                            ______________________________________                                    

whose pH was adjusted to 7.0. This was sterilized 15 minutes at 121° C.One liter of the second stage above was then charged and the mixture wasincubated at 85 rpm for 12 hours then 130 rpm for 84 hours at 28° C.with an air flow of 5 cfm for 12 hours then 10 cfm for 84 hours.

B. Direct Isolation of the Ammonium Salt of Compound II

The broth from the fermentation as in Example 7A (100 gal) is acidifiedwith H₃ PO₄ to pH of 5. Ethyl acetate (70 gal) is added and the mixtureis stirred vigorously. It is then filtered from the mycelia residue andthe cake is washed with a small amount of ethyl acetate which iscombined with the main extract. The organic phase is separated and mixedwith 5 gallons of 0.2 N sodium hydroxide solution. The mixture isstirred vigorously and then allowed to settle. The aqueous layer isseparated and the pH is adjusted from 9 to 5 by addition of H₃ PO₄. Itis then extracted, first with 2 gallons of hexane-ethyl acetate 2:1mixture and then with one gallon of the same mixture. The separateorganic extracts are combined and dried over anhydrous MgSO₄. The dryingagent is then separated by filtration and the cake washed with one literof the same hexane-ethyl acetate solution, the rinse being combined withthe filtrate. This filtrate solution, after further dilution with 2 L ofacetone, is stirred while ammonia gas is passed in. The gas is absorbedand a crystalline precipitate appears. When ammonia is no longerabsorbed, and a darkening in color is observed in the precipitate, theintroduction of ammonia is terminated and the mixture is allowed tostand several hours after which it is filtered. The crude ammonium saltfilter cake is washed with acetone to a colorless wash and is then airdried (128 g).

The crude ammonium salt can be recrystallized by dissolving it in amixture of chloroform, methanol and concentrated aqueous ammoniumhydroxide (80:20:2) and filtering the colored insoluble material (about10% of the crude). The filtrate solution is then diluted with an equalvolume of ether. The crystalline, tan colored ammonium salt is obtainedby filtration.

Alternatively, the 128 g of crude ammonium salt are suspended in 2liters toluene and heated to reflux under a water separator while a slowstream of nitrogen is passed through the solution. After about 21/2hours no more water is separated and the effluent gas contains no moreammonia. The hot solution of the lactone is filtered from about 10 g ofundissolved, dark material with the help of 13 g of activated charcoaland some Super-Cel. The filtrate is allowed to cool and kept at -20° C.overnight. The crystalline product is filtered, washed with cold tolueneand low boiling petrolether and air dried (76.6 g). From the combinedand concentrated mother liquor and washing solutions a second batch of17.4 g is obtained.

The product from this and a second similar batch (total 164.4 g) isrecrystallized from ethanol yielding 143.4 g of ≧99% pure compound inseveral batches.

71.5 G of recrystallized lactone is suspended in 200 ml of 1 N NaOH and200 ml CH₃ OH and stirred until all material is dissolved. The solutionis filtered, the methanol evaporated under reduced pressure at 30° C.and 200 ml ethylacetate and 200 ml 2 M H₃ PO₄ added. The aqueous phaseis saturated with NaCl and separated. The organic phase is dried overMgSO₄ and filtered and diluted to about 1 liter with ethylacetate andsome ether (filter washes). One liter of a mixture of chloroform,methanol and concentrated ammonium hydroxide (80:20:2), followed by 1liter of acetone are then added with stirring. The precipitated productis filtered and washed with acetone and low boiling petrolether and airdried (75.0 g). A second batch of 2.5 g is obtained from the combinedmother liquor and washing solution.

The final purification is achieved by dissolving 75 g of this ammoniumsalt in 2 liters of hot isopropanol containing 5% concentrated aqueousammonium hydroxide. The hot solution is rapidly filtered into apreheated flask and allowed to cool to room temperature after the excessof ammonia, which had been lost in the vacuum filtration, is replenishedby some NH₃ gas. Crystallization is completed by further cooling, firstto 0° C. and overnight to -20° C. The product is filtered and washedwith isopropanol and acetone at -20° C. and with low boiling petroletherand air dried under exclusion of moisture (65.2 g; ≧99.5% pure).

EXAMPLE 8 Salts of Compound II

To a solution of 40 mg of the product of Example 6 in 2 ml of ethanol isadded 1 ml of aqueous NaOH (10⁻⁴ moles; 1 equivalent). After one hour atroom temperature, the mixture is taken to dryness in vacuo to yield thesodium salt of Compound II.

In like manner the potassium salt is prepared using one equivalent ofpotassium hydroxide.

EXAMPLE 9 L-Lysine Salt of Compound II

A solution of 146 mg of L-lysine in 1.5 ml of 65% ethanol is added to asolution of 440 mg of the ammonium salt of Compound II in 11.5 ml of 85%ethanol. The solvents are distilled off in vacuo. The residue istriturated with 10 ml of warm ethanol, cooled, and filtered, and thewhite solid is dried to obtain 430 mg of the L-lysine salt of CompoundII, m.p. 178°-180°(d).

Anal. Calcd. for C₃₀ H₅₂ N₂ O₈ : C, 63.35; H, 9.22, N, 4.93; Found: C,62.80; H, 9.13; N, 4.83.

EXAMPLE 10 L-Arginine Salt of Compound II

In the manner substantially as described in Example 9, a solution of 174mg of L-arginine base and a solution of 440 mg of the ammonium salt ofCompound II are combined. The solvent is evaporated in vacuo, and theresidue is triturated with warm ethanol, cooled, filtered, and dried togive the L-arginine salt of Compound II.

EXAMPLE 11 L-Ornithine Salt of Compound II

In the manner substantially as described in Example 9, a solution of 132mg of L-ornithine free base and a solution of 440 mg of ammonium salt ofCompound II are combined. The solvent is evaporated in vacuo and theresidue is triturated with warm ethanol, cooled, filtered, and dried togive the L-ornithine salt of Compound II.

EXAMPLE 12 N-Methylglucamine Salt of Compound II

In the manner substantially as described in Example 9, a solution of 195mg of N-methylglucamine in 1.5 ml of water and 440 mg of Compound IIIammonium salt in 11.5 ml of 85% ethanol are combined. The solvent isevaporated in vacuo to obtain the N-methylglucamine salt of Compound II.

EXAMPLE 13 Ethylenediamine Salt of Compound II

18 g of Compound I are dissolved in 180 ml of warm isopropanol andtreated with 90 ml of 0.5 M aqueous NaOH solution, aged one hour,diluted with 180 ml of water, evaporated in vacuo to remove theisopropanol, and cooled in an ice bath. Slowly, 90 ml of 0.5 M HCl isadded and the mixture is extracted with 2×150 ml of ethyl acetate whichis backwashed with 100 ml of water, dried over MgSO₄. The solvent isremoved in vacuo at low temperature and the residue is dissolved in 150ml of ethanol. 3 Ml of ethylenediamine is added and the solvent isevaporated in vacuo, and the residue triturated with boiling ethylacetate, cooled, filtered, and recrystallized from 30 ml of isopropanoland dried in vacuo over P₂ O₅ to obtain 13.1 g of white crystals, m.p.152°-153.5° C.

Anal. Calcd. for (C₂₄ H₃₇ O₆)₂ ⁻.(C₂ H₁₀ N₂)⁺⁺ : C, 66.35; H, 9.35; N,3.09; Found: C, 66.08; H, 9.49; N, 3.01

EXAMPLE 14 Calcium Salt of Compound II

87.9 mg ammonium salt of Compound II are dissolved in 3 ml H₂ O withstirring and heating. 7.4 Mg analytical grade Ca(OH)₂ are then added andthe mixture stirred and heated until no more ammonia evaporates and onlya slight turbidity remains which is separated by centrifugation. Thecolorless, clear supernatant is lyophilized and probes of the drymaterial set up for crystallization from various solvents and solventmixtures. The product crystallizes in needles when a hot concentratedsolution in dry isopropanol is allowed to cool to room temperature.

EXAMPLE 15 Tetramethylammonium Salt of Compound II

34 Mg Compound I in 1 ml CH₂ Cl₂ are treated with 0.04 ml 24%tetramethylammonium hydroxide in methanol. The product is precipitatedwith ether in partially crystalline form, centrifuged and theprecipitate first washed with ether and then recrystallized as hexagonalplates from 1 ml of isopropanol by the addition of 5 ml ether and about5 ml low boiling petrol ether. 27 Mg or 65% are obtained.

    ______________________________________                                        .sup.1 H Nmr Spectrum of Compound II Tetramethylammonium                      Salt                                                                          ______________________________________                                        (6 mg/0.35 ml at 25° C. in CDCl.sub.3 at 300 MHz)                      0.83 t (3H, J = 6.5)                                                          0.84 d (3H, J = 7)                                                            1.02 d (3H, J = 7)                                                            1.05 d (3H, J = 7)                                                            1.24 m (˜1H); 1.30-1.80 br.m. envelope                                  1.88 ddd (1H, J = 2, 8, 15)                                                   1.98 dd (1H, 3, 15)                                                           2.16 dd (1H, J = 8.5, 15.5)                                                   2.23 m (1H, obscured)                                                         2.32 m (1H, obscured)                                                         2.37 dd (1H, J = 3, 15.5)                                                     2.40 m (˜1H, obscured)                                                  3.42 s (12H, MeN.sup.+)                                                       3.79 m (1H, symmetrical multiplet)                                            4.06 m (1H, symmetrical multiplet)                                            5.32 dt (1H, J ≅ 3)                                                 5.50 br.s (1H)                                                                5.79 dd (1H, J = 6, 10)                                                       5.98 d (1H, J = 10)                                                           ______________________________________                                    

Chemical shifts are in ppm downfield of internal TMS; coupling constantsin brackets are in Hz.

Abbreviations: s=singlet, d=doublet, t=triplet, m=multiplet

EXAMPLE 16 Ammonium Salt of Compound II

In the manner substantially as described in Example 13, Compound I isconverted to the hydroxy acid, Compound II, extracted into ethylacetate, dried over MgSO₄, filtered, and treated with anhydrous ammoniawith stirring and cooling to precipitate the ammonium salt.

EXAMPLE 17 Compound II

453 Mg of the ethylenediamine salt of Compound II are dissolved in 6 mlof 80% ethanol, cooled in an ice bath, treated with 1 ml of 1 M HCl,evaporated in vacuo to remove the ethanol, treated with 3 ml more water,extracted into 2×5 ml of ethyl acetate, and backwashed with water,keeping all solutions cold in an ice bath. The extract is dried overMgSO₄ and concentrated to dryness in vacuo to obtain the hydroxy acid asa colorless oil.

A ¹³ C-nmr spectrum in CDCl₃ (190 mg/ml) exhibits chemical shifts forthe first six carbons of the hydroxy acid moiety as listed in the table.Upon standing, this hydroxy acid slowly reverts to the lactone.

                  TABLE                                                           ______________________________________                                        .sup.13 C--Nmr Spectrum, Ppm Downfield from Tetramethylsilane                                    Hydroxy Acid,                                                                 Compound II                                                ______________________________________                                         ##STR5##        C.sub.1 C.sub.2, C.sub.4 C.sub.3 C.sub.5 C.sub.6                                      174.8 42.4, 41.6 68.8  72.3 34.9                     ______________________________________                                    

The spectrum of the remainder of the molecule is only slightly changedfrom the cyclized structure.

EXAMPLE 18 Ethyl Ester of Compound II

A suspension of 500 mg (1.24 mmol) of Compound I (MSD-803) in 20 ml ofethanol is stirred at room temperature under a nitrogen atmosphere. Asmall piece of sodium (Ca. 1 mg) is added. After 15 minutes a secondsmall piece of sodium is added. After a total reaction time of 30minutes the homogeneous reaction mixture is diluted with ether, washedwith water and with saturated brine and dried (MgSO₄). Evaporation ofthe solvent gives a waxy solid. Analysis by HPCL on a Whatman Partasil10 PAC column (4.6 mm×25 cm) with 10% isopropanol/hexane pumped at 6ml/min indicated a mixture of ethyl ester and MSD-803 (77:23). Thismixture is separated by medium-pressure chromatography on silica gel(230-400 mesh) by elution with 3% ethanol/methylene chloride. Thefractions containing the ester are combined and evaporated to give 358mg (66%) of an off-white solid, m.p. 67° C.

A portion of this material is recrystallized from hexane to give whiteneedles: m.p. 66.5°-68.5°.

Anal. Calc. for C₂₆ H₄₂ O₆ : C, 69.30; H, 9.40; Found: C, 69.22; H,9.58.

In like manner, by the use of equivalent amounts of methanol, propanol,butanol, isobutanol, t-butanol, amyl alcohol, isoamyl alcohol,2-dimethylaminoethanol, benzyl alcohol, phenethanol, 2-acetamidoethanoland the like, the corresponding esters are obtained.

EXAMPLE 19 Comparison of I and ML-236B as Inhibitors of Sterol Synthesisin Cell Culture

Employing the procedure of Example 1C, purified samples of Compound Iand ML-236B were shown to have IC₅₀ of 2.2 and 5.6 nM respectively asinhibitors of HMG-CoA reductase.

What is claimed is:
 1. A process for the isolation of the compound##STR6## as its ammonium salt from a fermentation broth obtained fromthe cultivation of microorganisms identified as Aspergillus terreus withAccession numbers ATCC 20541 and ATCC 20542 which comprises the stepsof:(a) acidifying whole broth; (b) extracting with a water-immiscible,inert, organic solvent; (c) extracting the organic solvent extract withaqueous alkali; (d) acidifying the aqueous extract; (e) extracting theaqueous extract with a water-immiscible, inert, organic solvent; (f)drying the organic solvent extract; and (g) treating the organic solventextract with gaseous ammonia.
 2. The process of claim 1 whereinacidification is with phosphoric acid to about pH 5 and the immiscibleorganic solvent is ethyl acetate or mixtures of n-hexane-ethyl acetate.